The Arabidopsis GORK K+-channel is phosphorylated by calcium-dependent protein kinase 21 (CPK21), which in turn is activated by 14-3-3 proteins.

Potassium (K+) is a vital ion for many processes in the plant and fine-tuned ion channels control the K+-fluxes across the plasma membrane. GORK is an outward-rectifying K+-channel with important functions in stomatal closure and in root K+-homeostasis. In this study, post-translational modification of the Arabidopsis GORK ion channel and its regulation by 14-3-3 proteins was investigated. To investigate the possible interaction between GORK and 14-3-3s an in vivo pull-down from an Arabidopsis protein extract with recombinant GORK C-terminus (GORK-C) indeed identified endogenous 14-3-3s (LAMBDA, CHI, NU) as binding partners in a phosphorylation dependent manner. However, a direct interaction between 14-3-3's and GORK-C could not be demonstrated. Since the pull-down of 14-3-3s was phosphorylation dependent, we determined GORK-C as substrate for CPK21 phosphorylation and identified three CPK21 phospho-sites in the GORK protein (T344, S518 and S649). Moreover, interaction of 14-3-3 to CPK21 strongly stimulates its kinase activity; an effect that can result in increased GORK phosphorylation and change in activity. Using the non-invasive vibrating probe technique, we measured the predominantly GORK mediated salt induced K+-efflux from wild-type, gork, cpk21, aha2 and 14-3-3 mutant roots. The mutants cpk21 and aha2 did not show statistical significant differences compared to WT. However, two (out of six) 14-3-3 isoforms, CHI and PHI, have a clear function in the salt induced K+-efflux. In conclusion, our results show that GORK can be phosphorylated by CPK21 and suggest that 14-3-3 proteins control GORK activity through binding with and activation of CPK21.

Higher order Arabidopsis 14-3-3 mutants show 14-3-3 involvement in primary root growth both under control and abiotic stress conditions.

Arabidopsis 14-3-3 proteins are a family of conserved proteins that interact with numerous partner proteins in a phospho-specific manner, and can affect the target proteins in a number of ways; e.g. modification of enzymatic activity. We isolated T-DNA insertion lines in six 14-3-3 genes within the non-epsilon group that phylogenetically group in three closely related gene pairs. In total, 6 single, 3 double, 12 triple, and 3 quadruple mutants were generated. The mutants were phenotyped for primary root growth on control plates: single and double mutants were indistinguishable from WT, whereas six triples and all quadruples showed a shorter primary root. In addition, length of the first epidermal cell with a visible root hair bulge (LEH) was used to determine primary root elongation on medium containing mannitol and 1-aminocyclopropane-1-carboxylic acid (ACC). This analysis showed clear differences depending on the stress and 14-3-3 gene combinations. Next to the phenotypic growth analyses, a 14-3-3 pull-down assay on roots treated with and without mannitol showed that mannitol stress strongly affects the 14-3-3 interactome. In conclusion, we show gene specificity and functional redundancy among 14-3-3 proteins in primary root elongation under control and under abiotic stress conditions and changes in the 14-3-3 interactome during the onset of stress adaptation.